表征PTEN:Ki67相互作用及其对胶质母细胞瘤放射抵抗性的贡献

Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults, comprising approximately 50% of newly diagnosed solid brain tumors. Newly diagnosed GBM patients have a poor prognosis and median survival of only 12-15 months with current standard of care therapy. Precision oncology approaches that have worked in other cancers have largely failed to translate to GBM due to several unique GBM properties, including resistance to cell death caused by DNA damaging agents such as radiation therapy. One novel mechanism of radioresistance is through enhanced DNA damage response (DDR) mediated by nuclear-localized tyrosine-phosphorylated PTEN (pY240-PTEN) recruited to chromatin through interaction with the PP1 Binding Domain (PP1BD) of Ki67. While PTEN is known to play an important tumor-suppressive role and is found to be altered in approximately 40% of GBM, an analysis of an EGFR-positive, PTEN-positive patient cohort revealed that the presence of pY240-PTEN correlated with worse overall survival. As a more complete understanding of this interaction may provide valuable insight as to the role that Ki67 plays in GBM radioresistance, N-terminal biotinylated peptides were utilized to examine the contribution of each amino acid of the Ki67-PP1BD to PTEN binding. Through streptavidin pulldown assays, we uncovered a basic 5-amino acid patch critical for PTEN:Ki67 binding and through phosphorylation of these synthetic peptides, determined that Ki67 pT525 may play a role in determining occupancy of the Ki67-PP1BD. To further investigate the impact of PTEN:Ki67 interaction-dependent DDR, a Ki67 KO TS528 glioma stem-like cell line was established. Characterization of this line indicated that Ki67 loss had little impact on unperturbed cells but following radiation, resulted in decreased proliferation, decreased DDR, and a more significant G2 cell cycle arrest compared to WT TS528. Additionally, in vivo radiation treatment of mice harboring TS528 Ki67 KO tumors imparted a significant increase in overall survival, while TS528 parental cells had no benefit. To determine the contribution of PTEN to the observed Ki67 KO phenotype, Ki67 was knocked out in PTEN-null U87 glioma cells. In concordance with previous findings of PTEN:Ki67 interactions following radiation, I found that Rad51 foci formation was increased only in U87 cells expressing both Ki67 and PTEN. The impact of PTEN binding was also examined in TS528 Ki67 KO cells through lentiviral restoration of Ki67 with minigene constructs expressing WT Ki67-PP1BD, or harboring mutations disrupting PTEN binding, fused to the chromatin binding LR domain. Alanine substitution of critical PTEN binding residues disrupted interaction by CoIP and is currently being tested for radiosensitizing effects in vivo . This work characterizes a novel DNA damage repair pathway implicated in GBM radioresistance and proposes a new potential target for therapeutic intervention.